Monocot seed product comprising a human blood protein

ABSTRACT

The invention is directed to blood proteins produced in monocot seeds and isolated therefrom for use in therapeutic compositions, as well as to methods of making these isolated blood proteins and to therapeutic compositions comprising them.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/751,869, filed Mar. 31, 2010, now issued as U.S. Pat. No.8,158,857, which is a continuation application of U.S. patentapplication Ser. No. 11/987,053, filed Nov. 27, 2007, now abandoned,which is a divisional of U.S. patent application Ser. No. 10/411,395,filed Apr. 11, 2003, now U.S. Pat. No. 7,304,208, which, in turn, is acontinuation-in-part of U.S. application Ser. No. 10/077,381, filed Feb.14, 2002, now U.S. Pat. No. 6,991,824, which claims priority benefit toU.S. provisional application Ser. No. 60/269,199, filed Feb. 14, 2001,application Ser. No. 10/077,381 being a continuation-in-part of U.S.patent application Ser. No. 09/847,232, filed May 2, 2001, nowabandoned, which claims priority benefit to U.S. provisional applicationSer. No. 60/266,920, filed Feb. 6, 2001, and U.S. provisionalapplication Ser. No. 60/201,182, filed May 2, 2000. All priorityapplications are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

A “Sequence Listing” is submitted with this application in the form of atext file, created 17 Apr. 2012, and named “506658027US04seqlist.txt”(22,510 bytes), the contents of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to human blood proteins produced in theseeds of monocot plants for use in making human and animal topicalcompositions and human therapeutic compositions.

BACKGROUND OF THE INVENTION

Many human blood proteins are in short or limited supply due to thelarger quantities required of the protein for positive therapeuticeffect or possibly also due to the larger demand of these proteins bythe world population of patients having the particular condition. It isalso advantageous to produce blood proteins, normally extracted fromblood products, from an alternative source such as crop plants.Production of blood proteins from plants mitigates contamination of theblood protein fraction with human viruses and other disease causativeagents found in human or animal blood product fractions.

Blood proteins such as hemoglobin, alpha-1-antitrypsin (“AAT”),fibrinogen, human serum albumin, thrombin, antithrombin III, antibodies,blood coagulation factors (e.g. Factors V-XIII), and others are known tohave therapeutic potential for a number of human conditions.

Hemoglobin is the major blood component molecule transporting oxygen tocells. Mammmalian hemoglobins are tetrameric proteins made up of twoex-like polypeptide subunits and two non-α (usually β, γ, or δ)subunits. These subunits differ in primary amino acid sequence, but havesimilar secondary and tertiary structures. Each globin subunit hasassociated with it, by noncovalent interaction, a Fe²+-porphyrin complexknown as a heme group, to which oxygen binds. The predominant hemoglobinin adult erythrocytes is α2β2, known as hemoglobin A₁ (HbA). Eachhemoglobin tetramer has a molecular weight of 64 kD and each α-like andβ-like chain has a molecular weight of approximately 15.7 kD (141 aminoacids) and 16.5 kD (146 amino acids) respectively.

AAT belongs to the class of serpin inhibitors and is one of the majorprotease inhibitors in human plasma. AAT is a single 394 amino acidpolypeptide having an approximate molecular mass of 52 kD, and containsabout 15% carbohydrate in the native human form of the molecule.Concentrations of AAT in human plasma range from 1000-3000 mg/L and inhuman milk range from 100 to 400 mg/L Its primary physiological role isthe inhibition neutrophil elastase, with an insufficiency leading to thedevelopment of pulmonary emphysema. Excess production of elastaseactivity leads to emphysema, hepatitis and a variety of skin disorders.While the binding affinity of AAT is highest for human neutrophilelastase, it also has affinity for pancreatic proteases such aschymotrypsin and trypsin. The current primary source for the treatmentof AAT deficiency is isolating AAT from human blood plasma.

Fibrinogen is involved in the blood coagulation cascade and is convertedto fibrin by its interaction with the natural clotting agent thrombin.Fibrin is the major component of blood clotting. Mature human fibrinogenconsists of two pairs of three independent polypeptide chains (α, β andγ) that are linked together by 29 intra- and intermolecular disulfidebonds forming a native protein of 340 kD and is present in human plasmaat an approximate concentration of 2500 mg/L. Three-dimensionalstructural analysis of independent fibrinogen domains has provideddetailed structural features giving important clues to humanfibrinogen's multifunctional role. The fibrinogen polypeptides areapproximately 72 kD (α), 52 kD (β) and 48 kD (γ) respectively with the βpolypeptide chain determining native molecule assembly. The structure offibrinogen features a number of structural and functional domainscontaining multiple binding sites that facilitate interactions withitself, other proteins, certain cell types and allow fibrinogen toparticipate in a number of important physiological processes includingblood coagulation, inflammation, angiogenesis, wound closure,artheriogenesis and tumorigenesis. Fibrin formation from a clottingstandpoint is mediated by the interaction of native fibrinogen with itsnatural clotting agents Factor XIII and thrombin in the presence ofblood soluble calcium.

Albumin is a transport protein molecule that carries out many functionsin mammalian serum biology, notably that of a carrier of hormones andother soluble ligands from site to site, and other activities thatcontribute largely to general mammalian biochemistry. Human serumalbumin (“HSA”) is also the major protein component of blood beingactively present at serum concentrations of approximately 30,000-50,000mg/L. HSA is a single polypeptide chain of 66.5 kD that is initiallysynthesized as a prepro-albumin molecule in the liver and released fromthe endoplasmic reticulum after N-terminal and C-terminal Golgiprocessing. The resultant mature protein is 585 amino acids in length.It has been shown that the natural preprosequnce of HSA can function incorrect protein targeting/processing across a plant plasma membrane intransgenic tobacco leaves (Sijmons et al., 1990).

Prothrombin, a plasma glycoprotein, is the zymogen of the serineprotease thrombin that catalyzes the conversion of fibrinogen to fibrinas well as several other reactions that may be important for bloodcoagulation. Prothrombin is a single polypeptide chain approximately72,000 molecular weight in size. The complete human thrombin cDNAconsists of 622 amino acid residues and includes a leader sequence of 36amino acid residues. Active thrombin has an apparent molecular weight of36,000 and is made up of two disulfide-linked polypeptide chainsresulting from prothrombin cleavage. The proteolytic events leading toin vitro activation and conversion of human prothrombin to activethrombin have been extremely well characterized. Antithrombin III is asingle chain glycoprotein with a molecular weight of 58 kD. It is amember of the serpin (serine protease inhibitor) super family and isconsidered to be the most important inhibitor in the coagulationcascade. Antithrombin III inhibits a wide spectrum of serine proteasesincluding thrombin, factors IXa, Xa and XIa, kallikrein, plasmin,urokinase, C1-esterase, and trypsin. Antithrombin III activity ismarkedly potentiated by heparin; potentiation of its activity is theprinciple mechanism by which both heparin and low-molecular-weightheparin produce anticoagulation.

Factors V-XIII are proteins (mostly proteases in their active states)that are involved in the ‘intrinsic pathway’ of the classical casademechanism for blood coagulation. The majority of these molecules existas precursors that are processed in an ordered sequence oftransformations from inactive to catalytically active forms. Factor V isproaccelerin (the accelerator globulin) while Factor VI is the activatedform of Factor V. Factor VII is proconvertin, the plasma thromboplastincomponent, while Factors VIII (antihemophilic factor) and IX (Christmasantihemophilic factor) are both associated with the hemophilia diseasestate. Factors X (Stuart-Power factor), XI (plasma thromboplastinanticedent) and XII (Hageman factor) are all involved with thematuration/stabilization of thrombin. Factor XIII (fibrin stabilizingfactor) is a plasma transglutaminase directly acting on fibrin duringthe clotting process. All these Factors are present at relatively low inserum plasma (0.001 to 50 mg/L). Other protein factors also involved inthe blood coagulation cascade include Fletcher Factor (prekallikrein),Fitzgerald factor (kininogen) and von Willebrand Factor.

Immunoglobulins (antibodies) present in humans act to confer resistanceto a variety of pathogens to which a patient may have been exposed.Immunoglobulin molecules account for 15-20% of the mass in human serumand consist predominantly of IgG, IgM and IgA-type antibodies involvedin fighting various infections that invade the blood system andpotentially the rest of the body. IgG type antibodies are the mostprevalant and exist at a serum concentration of between 618 g/L. Theblood system also serves as a carrier directing these molecules tospecific areas of the body to combat resulting infections and potentialoncogenic targets. Mature antibodies consist of two polypeptides (lightand heavy chains) that must be expressed in eqimolar amounts and cometogether to form functional entities. The light chain (˜25 kD) is aprotein of ˜210-240 amino acids in length while the heavy chain (˜50 kD)is a protein of ˜450-460 amino acids in length. Both light and heavychains carry signal peptides for processing and secretion into the bloodstream. Expression of monoclonal antibodies in plants is of particularinterest, because it requires the expression of two genes, synthesis oftwo proteins and correct assembly of the tetrameric protein to result ina functional antibody.

Initial studies of antibodies in plants focused on the IgG antibodyclass (Hiatt et al., 1989; Hiatt and Ma, 1992), but later studiesexplored the in planta expression of complex antibody molecules such assecretory IgA antibodies (4 genes) and more complex antibody forms (Maet al., 1995; Vine et al., 2001).

US. Pat. Nos. 6,471,429, 5,959,177, 5,639,947 and 5,202,422, all relatedpatents, disclose the production of antibody molecules in transgenictobacco plant leaves.

U.S. Pat. No. 6,303,341 discloses the production of immunoglobulinscontaining protection proteins in tobacco plant leaves, stems, flowersand roots.

Published U.S. Patent Application U.S. 2002/0174453 discloses theproduction of antibodies in the plastids of tobacco plants.

Published U.S. Patent Application U.S. 2002/0046418 discloses acontrolled environment agriculture bioreactor for the commercialproduction of heterologous proteins in transgenic plants. Thespecification discloses that production of mammalian blood proteins canbe achieved. Example 7 discloses the production of human blood factorsin the leaves of potato, tobacco and alfalfa plants.

U.S. Pat. No. 6,344,600 discloses the production of hemoglobin andmyoglobin in tobacco plant leaves. Example X discloses the extractionand partial purification of recombinant hemoglobin from tobacco seeds.The expression was obtained by transformation of the coexpressionplasmid pBIOC59, which was constructed to allow targeting in thechloroplasts, and contained for this purpose the transit peptide of theprecursor of the small subunit of ribulose 1,5-diphosphate carboxylaseof Pisum sativum L. Expression in seeds was reported to be at a maximumlevel of 0.05% recombinant hemoglobin relative to the total solubleproteins extracted.

Example XI of the '600 patent discloses the construction of plasmidscontaining one of the α or β chains of hemoglobin allowing constitutiveexpression or expression in the albumin in maize seeds. According tothis disclosure, the constitutive or albumin-specific expression of thehemoglobin chains required the following regulatory sequences: one ofthree promoters allowing a constitutive expression ((i) the rice actinpromoter followed by the rice actin intron, contained in the plasmidpAct1-F4; (ii) the 35S double constitutive promoter of cauliflowermosaic virus; or (iii) the promoter of the maize γ-zein gene containedin the plasmid pγ63) and one of two terminators ((i) the 35S polyAterminator; or (ii) the NOS polyA terminator). No experiment or data isprovided regarding transformation or expression of these plasmids inmaize or maize seeds.

U.S. Pat. No. 5,767,363 discloses the use of a seed-specific promoterderived from ACP of Brassica napus, to affect and vary the expression ofseed oils in rape and tobacco plants. The specification genericallydiscloses that the seed specific promoter can be used for the expressionof pharmaceutical proteins, such as blood factors or human serumalbumin, however no experimental data whatsoever is presented in thisregard.

Daniell et al. (2001) is a review article discussing recent developmentsin the field of medical molecular farming, including the production ofantibodies and proteins in plants.

None of these patents or publications discloses the production of humanblood proteins in monocot seeds in high yield. It is desirable toprovide for the production of human blood proteins in high yield freefrom contaminating source agents in order to provide the patientpopulation with sufficient supply of these proteins for use in treatinghumans with conditions treatable by administration of a particular bloodprotein.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a method of producing arecombinant human blood protein in monocot plant seeds, comprising thesteps of:

-   -   a. transforming a monocot plant cell with a chimeric gene        comprising a promoter from the gene of a maturation-specific        monocot plant storage protein,        -   i. (ii) a first DNA sequence, operably linked to said            promoter, encoding a monocot plant seed-specific signal            sequence capable of targeting a polypeptide linked thereto            to a monocot plant seed endosperm cell, and        -   ii. (iii) a second DNA sequence, linked in translation frame            with the first DNA sequence, encoding a human blood protein,            wherein the first DNA sequence and the second DNA sequence            together encode a fusion protein comprising an N-terminal            signal sequence and the human blood protein;    -   b. growing a monocot plant from the transformed monocot plant        cell for a time sufficient to produce seeds containing the human        blood protein; and    -   c. harvesting the seeds from the plant, wherein the human blood        protein constitutes at least 3.0% of the total soluble protein        in the harvested seeds.

The invention also includes a purified human blood protein obtained bythe method. Preferably, the human blood protein comprises one or moreplant glycosyl groups.

The invention also provides a monocot plant seed product, preferablyselected from whole seed, flour, extract and malt, prepared from theharvested seeds obtained by the method of the invention. Preferably, thehuman blood protein constitutes at least 3.0% of the total solubleprotein in the seed product.

The invention further provides a composition comprising a purified humanblood protein, preferably comprising at least one plant glycosyl group,and at least one pharmaceutically acceptable excipient or nutrient,wherein the human blood protein is produced in a monocot plantcontaining a nucleic acid sequence encoding the human blood protein andis purified from seed harvested from the monocot plant. The nutrient isfrom a source other than the monocot plant. The formulation can be usedfor parenteral, enteric, inhalation, intranasal or topical delivery.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows plasmids with constructs containing three codon-optimizedgenes encoding the fibrinogen polypeptides α (pAPI 398), β (pAP I 417)and γ (pAPI 327) (SEQ ID NO: 1-3), each under the control of the riceglutelin promoter Gt1. These plasmids, including a plasmid (not shown)containing the hygromycin selectable marker, were bombarded intoembryogenic rice callus to create transgenic rice plants expressingthese three genes in mature rice seeds.

FIG. 2 shows a Western blot analysis of transgenic rice lines expressingindividual subunits of human fibrinogen. Lane 1, positive control,purified native human fibrinogen (obtained from the Red Cross) showingall three polypeptide chains; Lane 2, extract from Tapei 309, a nontransgenic rice variety; Lane 3, molecular weight standard; Lane 4, riceseed extract expressing fibrinogen a chain; Lane 5, rice seed extractexpressing fibrinogen β chain; Lane 6, rice seed extract expressingfibrinogen γ chain. Total protein extract of rice seeds was performed in2% SDS, 1M urea, 1% βMe and PBS pH 7.4. Fibrinogen polypeptides weredetected using antibody recognizing all three chains or individualchains only.

FIG. 3 shows the simultaneous expression of the three fibrinogenpolypeptide chains (α, β and γ) in transgenic rice seeds and analyzedvia Western blot analysis. Fibrinogen polypeptides and proteinaggregates were detected Using antibody recognizing all three chains.FIG. 3A indicates total protein extracted from rice seeds undernon-denaturing conditions (350 mM NaCl, PBS pH 7.4, 0.01%Tween-20/Trition X-100/CHAPS) and run on a non-denaturing 10% acrylamidegel. Lane 1, 1 μg purified human fibrinogen; Lanes 2 & 3, extracts fromTapei 309, a nontransgenic rice variety; Lane 4, molecular weightmarkers; Lanes 5 & 7, extracts from two transgenic rice lines where 1.0%βMe was included in the extraction buffer; lanes 6 & 8, extracts fromtwo transgenic rice lines without βMe in the extraction buffer. Lanes 6& 8 show large protein aggregates that were extracted undernondenaturing conditions from the transgenic lines that run at theapproximate position of complexed native human fibrinogen. FIG. 3Bindicates total protein extracted from rice seeds in 2% SDS, 1M urea, 1%βMe and PBS pH 7.4, and run on SDS PAGE. Lane 1, positive control,native human fibrinogen (obtained from the Red Cross) showing all threepolypeptide chains; Lane 2, molecular weight standards; Lanes 3-5, threeindependent transgenic rice lines expressing all three fibrinigenpolypeptides.

FIG. 4 shows the plasmid pAPI 250 expressing the codon-optimized genefor alpha-1-antitrypsin (AAT) (SEQ ID NO:5) under the control of therice glutelin promoter Gt1. This plasmid, along with a plasmid (notshown) containing the hygromycin selectable marker gene, was bombardedinto embryogenic rice callus to create transgenic rice plants expressingAAT in mature rice seeds.

FIG. 5 shows Coomassie brilliant blue staining of aqueous phaseextraction of transgenic rice grains expressing human recombinant AAT.Both untransformed (rice var. Kitaake) and transgenic rice seeds (˜10pooled R1 seed from five individual transgenic plants) were ground withPBS pH 7.4 buffer. The resulting extract was spun at 14,000 rpm at 4° C.for 10 min. Supernatant was collected and ˜20 μg of this soluble proteinextract was resuspended in sample loading buffer, and loaded onto aprecast SDS-PAGE gel. Lane 1, molecular weight protein markers; Lane 2,purified non-recombinant human AAT; Lane 3, extract from controlnon-transformed Kitaake variety. Between lanes 2 and 3, the results fromthe extracts of the five individual transgenic plants are shown.

FIG. 6 shows Western blot analysis of recombinant human AA T expressedin transgenic rice grains. The R1 pooled seed soluble protein extracts(˜10 μg total protein) from seven independent transgenic ricetransformants were prepared as described in FIG. 5 above, separated bySDS-PAGE gel and then blotted onto a nitrocellulose filter. Theidentification of AAT expressed in rice seeds was carried out by Westernanalysis using anti-AAT antibody. Lane 1, molecular weight proteinmarkers; Lanes 2 & 3, 1 μg and 2 μg, respectively, of purifiednonrecombinant human AAT; Lanes 4 & 5, control, non-transgenic riceextract (var. Kitaake). The final seven lanes show the results from theextracts of the seven individual transgenic plants. Extracts from two ofthe seven transgenic lines did not express AAT. The shift in gelmobility between the non-recombinant human and recombinantrice-expressed forms is due to the type and glycosylation differences inthe human and recombinant rice-expressed proteins.

FIG. 7 shows activity of purified recombinant AAT (rAAT) obtained fromrice extracts against purified porcine pancreatic elastase (PPE) asdetermined by Coomassie staining and Western blot analysis. The activityof rAAT is demonstrated by a band shift assay involving the AAT proteasesubstrate, elastase. AAT samples from human and rice extracts wereincubated with equal number of moles of PPE at 37° C. for 15 min.Negative control for band shift assay was prepared with the AAT samplesincubated with equal volume of PPE added. Lane MW refers to molecularweight markers. Panel 7A: Lane 1, purified non-recombinant AAT fromhuman plasma; Lane 2, purified AAT from human plasma+PPE; Lane 3,soluble protein extract containing AAT from transgenic rice seed; Lane4, protein extract containing AAT from transgenic rice seed+PPE; Lane 5,non-transformed rice seed extract; Lane 6, non-transformed rice seedextract+PPE. Panel 7B shows a shifted band in Lanes 1, 2 and 3. Theshifted band, a complex between PPE and an AAT fragment is confirmed tocontain AAT by Western blot analysis. The lanes in Panel 7B areanalogous to those in Panel 7A.

FIG. 8 depicts AAT derived from rice cell extracts purified initiallythrough Con-A and DEAE Sepharose respectively, then loaded onto an octylSepharose column. Octyl Sepharose is the final purification step andseparates active AAT from an inactivated form of the protein. Lane 1,molecular weight markers; Lane 2, 2 μg purified non-recombinant humanAAT as a standard; Lane 3, pooled eluate from the DEAE Sepharose column.The remaining columns show the flow-through and the eluate from theoctyl Sepharose column. Approximately 50 μL from each column fractionwas loaded onto an SDS-PAGE gel and the proteins visualized by Coomassiestaining. Octyl Sepharose flow-through shows the inactive AAT proteinwhile the eluate resolves active AAT.

FIG. 9A depicts an AAT association rate constant for activity ofpurified recombinant AAT against PPE determined (as described by theprocedure in FIG. 7) using non-recombinant human AAT as a control. Datawere generated by Coomassie protein staining and Western blot analysis,as described in FIG. 7. FIG. 9B depicts the thermostability ofplant-derived recombinant AAT versus native human AAT determined by thePPE inhibition assay.

FIG. 10 shows the plasmid pAPI 9 for expression of codon-optimized humanserum albumin (HSA) (SEQ ID NO:4) under the control of the rice Amy1Apromoter/signal peptide. This plasmid is useful for the expression ofHSA in germinated rice seeds.

FIG. 11 shows the expression of HSA in transgenic rice seeds. Pooledseed from transgenic rice line 3-11-2 were imbibed in water for 24hours, then 2 μM gibberelic acid (GA) was added. Seed samples wereextracted at 24, 48, 72, and 120 hours post GA addition and solubleproteins were extracted and prepared for Westem analysis. 15 μg ofsoluble protein were loaded onto each lane along with protein isolatedfrom the non-transfromed negative control line TP309. The blot wasprobed with monoclonal antibody prepared against HSA.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, all terms used herein have the meaningsgiven below or are generally consistent with same meaning that the termshave to those skilled in the art of the present invention. Practitionersare particularly directed to Sam brook et al. (1989) Molecular Cloning:A Laboratory Manual (Second Edition), Cold Spring Harbor Press,Plainview, N.Y., Ausubel F. M. et al., (1993) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y., and Gelvin andSchilperoot , eds. (1997) Plant Molecular Biology Manual, KluwerAcademic Publishers, The Netherlands for definitions and terms of theart.

The polynucleotides of the invention may be in the form of RNA or in theform of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, andgenomic DNA The DNA may be double-stranded or single-stranded, and ifsingle-stranded may be the coding strand or the non-coding (anti-sense,complementary) strand.

The term “stably transformed” with reference to a plant cell means theplant cell has a non-native (heterologous) nucleic acid sequenceintegrated into its genome which is maintained through two or moregenerations.

By “host cell” is meant a cell containing a vector and supporting thereplication and/or transcription and/or expression of the heterologousnucleic acid sequence. Preferably, according to the invention, the hostcell is a monocot plant cell. Other host cells may be used as secondaryhosts, including bacterial, yeast, insect, amphibian or mammalian cells,to move DNA to a desired plant host cell.

A “plant cell” refers to any cell derived from a plant, includingundifferentiated tissue (e.g., callus) as well as plant seeds, pollen,propagules, embryos, suspension cultures, meristematic regions, leaves,roots, shoots, gametophytes, sporophytes and microspores.

The term “mature plant” refers to a fully differentiated plant.

The term “seed product” includes, but is not limited to, seed fractionssuch as de-hulled whole seed, flour (seed that has been de-hulled bymilling and ground into a powder) a seed extract, preferably a proteinextract (where the protein fraction of the flour has been separated fromthe carbohydrate fraction), malt (including malt extract or malt syrup)and/or a purified protein fraction derived from the transgenic grain.

The term “biological activity” refers to any biological activitytypically attributed to that protein by those of skill in the art.

The term “blood protein” refers to one or more proteins, or biologicallyactive fragments thereof, found in normal human blood, including,without limitation, hemoglobin, alpha-1-antitrypsin, fibrinogen, humanserum albumin, prothrombin/thrombin, antithrombin III, antibodies, bloodcoagulation factors (Factor V, Factor VI, Factor VII, Factor VIII,Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, FletcherFactor, Fitzgerald Factor and von Willebrand Factor), and biologicallyactive fragments thereof.

The term “non-nutritional” refers to a pharmaceutically acceptableexcipient which does not as its primary effect provide nutrition to therecipient. Preferably, it may provide one of the following services toan enterically delivered formulation, including acting as a carrier fora therapeutic protein, protecting the protein from acids in thedigestive tract, providing a time-release of the active ingredientsbeing delivered, or otherwise providing a useful quality to theformulation in order to administer to the patient the blood proteins.

“Monocot seed components” refers to carbohydrate, protein, and lipidcomponents extractable from monocot seeds, typically mature monocotseeds.

“Seed maturation” refers to the period starting with fertilization inwhich metabolizable reserves, e.g., sugars, oligosaccharides, starch,phenolics, amino acids, and proteins, are deposited, with and withoutvacuole targeting, to various tissues in the seed (grain), e.g.,endosperm, testa, aleurone layer, and scutellar epithelium, leading tograin enlargement, grain filling, and ending with grain desiccation.

“Maturation-specific protein promoter” refers to a promoter exhibitingsubstantially upregulated activity (greater than 25%) during seedmaturation.

“Heterologous DNA” refers to DNA which has been introduced into plantcells from another source, or which is from a plant source, includingthe same plant source, but which is under the control of a promoter thatdoes not normally regulate expression of the heterologous DNA.

“Heterologous protein” is a protein encoded by a heterologous DNA.

A “signal sequence” is an N- or C-terminal polypeptide sequence which iseffective to localize the peptide or protein to which it is attached toa selected intracellular or extracellular region. Preferably, accordingto the invention, the signal sequence targets the attached peptide orprotein to a location such as an endosperm cell, more preferably anendosperm-cell organelle, such as an intracellular vacuole or otherprotein storage body, chloroplast, mitochondria, or endoplasmicreticulum, or extracellular space, following secretion from the hostcell.

Expression vectors for use in the present invention are chimeric nucleicacid constructs (or expression vectors or cassettes), designed foroperation in plants, with associated upstream and downstream sequences.

In general, expression vectors for use in practicing the inventioninclude the following operably linked components that constitute achimeric gene: a promoter from the gene of a maturation-specific monocotplant storage protein, a first DNA sequence, operably linked to thepromoter, encoding a monocot plant seed-specific signal sequence (suchas an N-terminal leader sequence or a C-terminal trailer sequence)capable of targeting a polypeptide linked thereto to an endosperm cell,preferably an endosperm-cell organelle, such as a protein storage body,and a second DNA sequence, linked in translation frame with the firstDNA sequence, encoding a human blood protein. The Signal sequence ispreferably cleaved from the human blood protein in the plant cell.

The chimeric gene, in turn, is typically placed in a suitableplant-transformation vector having (i) companion sequences upstreamand/or downstream of the chimeric gene which are of plasmid or viralorigin and provide necessary characteristics to the vector to permit thevector to move DNA from bacteria to the desired plant host; (ii) aselectable marker sequence; and (iii) a transcriptional terminationregion generally at the opposite end of the vector from thetranscription initiation regulatory region.

Numerous types of appropriate expression vectors, and suitableregulatory sequences are known in the art for a variety of plant hostcells. The promoter region is chosen to be regulated in a mannerallowing for induction under seed-maturation conditions. In one aspectof this embodiment of the invention, the expression construct includes apromoter which exhibits specifically upregulated activity during seedmaturation. Promoters for use in the invention are typically derivedfrom cereals such as rice, barley, wheat, oat, rye, corn, millet,triticale or sorghum. Examples of such promoters include thematuration-specific promoter region associated with one of the followingmaturation-specific monocot plant storage proteins: rice glutelins,oryzins, and prolamines, barley hordeins, wheat gliadins and glutelins,maize zeins and glutelins, oat glutelins, and sorghum kafirins, milletpennisetins, and rye secalins. Exemplary regulatory regions from thesegenes are exemplified by SEQ ID NOS:6-14. Other promoters suitable forexpression in maturing seeds include the barley endosperm-specificB1-hordein promoter, GluB-2 promoter, Bx7 promoter, Gt3 promoter, GluB-1promoter and Rp-6 promoter, particularly if these promoters are used inconjunction with transcription factors.

Of particular interest is the expression of the nucleic acid encoding ahuman blood protein from a promoter that is preferentially expressed inplant seed tissue. Examples of such promoter sequences include thosesequences derived from sequences encoding plant storage protein genes orfrom genes involved in fatty acid biosynthesis in oilseeds. Exemplarypreferred promoters include a glutelin (Gt1) promoter, as exemplified bySEQ ID NO:6, which effects gene expression in the outer layer of theendosperm, and a globulin (Gib) promoter, as exemplified by SEQ ID NO:7,which effects gene expression in the center of the endosperm. Promotersequences for regulating transcription of gene coding sequences operablylinked thereto include naturally-occurring promoters, or regions thereofcapable of directing seed-specific transcription, and hybrid promoters,which combine elements of more than one promoter. Methods forconstruction such hybrid promoters are well known in the art.

In some cases, the promoter is native to the same plant species as theplant cells into which the chimeric nucleic acid construct is to beintroduced. In other embodiments, the promoter is heterologous to theplant host cell.

Alternatively, a seed-specific promoter from one type of monocot may beused regulate transcription of a nucleic acid coding sequence from adifferent monocot or a non-cereal monocot.

In addition to encoding the protein of interest, the expression cassetteor heterologous nucleic acid construct includes DNA encoding a signalpeptide that allows processing and translocation of the protein, asappropriate. Exemplary signal sequences are those sequences associatedwith the monocot maturation-specific genes: glutelins, prolamines,hordeins, gliadins, glutenins, zeins, albumin, globulin, AOP glucosepyrophosphorylase, starch synthase, branching enzyme, Em, and lea.Exemplary sequences encoding a signal peptide for a protein storage bodyare identified herein as SEQ ID NOS:15-21.

In one preferred embodiment, the method is directed toward thelocalization of proteins in an endosperm cell, preferably anendosperm-cell organelle, such as a protein storage body, mitochondrion,endoplasmic reticulum, vacuole, chloroplast or other plastidiccompartment. For example, when proteins are targeted to plastids, suchas chloroplasts, in order for expression to take place the constructalso employs the use of sequences to direct the gene product to theplastid. Such sequences are referred to herein as chloroplast transitpeptides (CTP) or plastid transit peptides (PTP). In this manner, whenthe gene of interest is not directly inserted into the plastid, theexpression construct additionally contains a gene encoding a transitpeptide to direct the gene of interest to the plastid. The chloroplasttransit peptides may be derived from the gene of interest, or may bederived from a heterologous sequence having a CTP. Such transit peptidesare known in the art. See, for example, (Smeekens et al., 1986; Wasmannet al., 1986; Von Heijne et at, 1991, U.S. Pat. Nos. 4,940,835 and5,728,925. Additional transit peptides for the translocation of theprotein to the endoplasmiC reticulum (ER) (Chrispeels, 1991; Vitale andChrispeels, 1992), nuclear localization signals (Shieh et al., 1993;Varagona et al., 1992) or vacuole (Raikhel and Chrispeels 1992; Bednarekand Raikel, 1992; also see U.S. Pat. No. 5,360,726) may also find use inthe constructs of the present invention.

Another exemplary class of signal sequences are sequences effective topromote secretion of heterologous protein from aleurone cells duringseed germination, including the signal sequences associated withalpha-amylase, protease, carboxypeptidase, endoprotease, ribonuclease,DNase/RNase, (1-3)-betaglucanase, (1-3)(1-4)-beta-glucanase, esterase,acid phosphatase, pentosamine, endoxylanase, β-xylopyranosidase,arabinofuranosidase, beta-glucosidase, (1-6)-beta-glucanase,perioxidase, and lysophospholipase.

Since many protein storage proteins are under the control of amaturation-specific promoter, and this promoter is operably linked to asignal sequence for targeting to a protein body, the promoter and signalsequence can be isolated from a single protein-storage gene, thenoperably linked to a blood protein in the chimeric gene construction.One preferred and exemplary promoter-signal sequence is from the riceGt1 gene, having an exemplary sequence identified by SEQ ID NO:6.Alternatively, the promoter and leader sequence may be derived fromdifferent genes. One preferred and exemplary promoter-signal sequencecombination is the rice Glb promoter linked to the rice Gt1 leadersequence, as exemplified by SEQ ID NO:7.

Preferably, expression vectors or heterologous nucleic acid constructsdesigned for operation in plants comprise companion sequences upstreamand downstream to the expression cassette. The companion sequences areof plasmid or viral origin and provide necessary characteristics to thevector to permit the vector to move DNA from a secondary host to theplant host, such as, sequences containing an origin of replication and aselectable marker. Typical secondary hosts include bacteria and yeast.

In one embodiment, the secondary host is E. coli, the origin ofreplication is a ColE1-type, and the selectable marker is a geneencoding ampicillin resistance. Such sequences are well known in the artand are commercially available as well (e.g., Clontech, Palo Alto,Calif.; Stratagene, La Jolla, Calif.

The transcription termination region may be taken from a gene where itis normally associated with the transcriptional initiation region or maybe taken from a different gene. Exemplary transcriptional terminationregions include the NOS terminator from Agrobacterium Ti plasmid and therice α-amylase terminator.

Polyadenylation tails may also be added to the expression cassette tooptimize high levels of transcription and proper transcriptiontermination, respectively. Polyadenylation sequences include, but arenot limited to, the Agrobacterium octopine synthetase signal, or thenopaline synthase of the same species.

Suitable selectable markers for selection in plant cells include, butare not limited to, antibiotic resistance genes, such as, kanamycin(nptll), G418, bleomycin, hygromycin, chloramphenicol, ampicillin,tetracycline, and the like. Additional selectable markers include a bargene which codes for bialaphos resistance; a mutant EPSP synthase genewhich encodes glyphosate resistance; a nitrilase gene which confersresistance to bromoxynil; a mutant acetolactate synthase gene (ALS)which confers imidazolinone or sulphonylurea resistance; and amethotrexate resistant DHFR gene.

The particular marker gene employed is one which allows for selection oftransformed cells as compared to cells lacking the DNA which has beenintroduced. Preferably, the selectable marker gene is one whichfacilitates selection at the tissue culture stage, e.g., a kanamyacin,hygromycin or ampicillin resistance gene.

The vectors of the present invention may also be modified to includeintermediate plant transformation plasmids that contain a region ofhomology to an Agrobacterium tumefaciens vector, a T-DNA border regionfrom Agrobacterium tumefaciens, and chimeric genes or expressioncassettes (described above). Further, the vectors of the invention maycomprise a disarmed plant tumor inducing plasmid of Agrobacteriumtumefaciens.

In general, a selected nucleic acid sequence is inserted into anappropriate restriction endonuclease site or sites in the vector.Standard methods for cutting, ligating and transformation into asecondary host cell, known to those of skill in the art, are used inconstructing vectors for use in the present invention. (See generally,Maniatis et al., Ausubel et al., and Gelvin et al., supra.)

Plant cells or tissues are transformed with expression constructs(heterologous nucleic acid constructs, e.g., plasmid DNA into which thegene of interest has been inserted) using a variety of standardtechniques. Effective introduction of vectors in order to facilitateenhanced plant gene expression is an important aspect of the invention.It is preferred that the vector sequences be stably transformed,preferably integrated into the host genome.

The method used for transformation of host plant cells is not criticalto the present invention. The skilled artisan will recognize that a widevariety of transformation techniques exist in the art, and newtechniques are continually becoming available. Any technique that issuitable for the target host plant may be employed within the scope ofthe present invention. For example, the constructs can be introduced ina variety of forms including, but not limited to, as a strand of DNA, ina plasmid, or in an artificial chromosome. The introduction of theconstructs into the target plant cells can be accomplished by a varietyof techniques, including, but not limited to calcium-phosphate-DNAco-precipitation, electroporation, microinjection,Agrobacterium-mediated transformation, liposome-mediated transformation,protoplast fusion or microprojectile bombardment (Christou, 1992;Sanford et al., 1993). The skilled artisan can refer to the literaturefor details and select suitable techniques for use in the methods of thepresent invention.

When Agrobacterium is used for plant cell transformation, a vector isintroduced into the Agrobacterium host for homologous recombination withT-DNA or the Ti-or Ri-plasmid present in the Agrobacterium host TheTi-or Ri-plasmid containing the T-DNA for recombination may be armed(capable of causing gall formation) or disarmed (incapable of causinggall formation), the latter being permissible, so long as the vir genesare present in the transformed Agrobacterium host The armed plasmid cangive a mixture of normal plant cells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) is inserted into a broad hostrange vector capable of replication in E. coli and Agrobacterium,examples of which are described in the literature, for example pRK2 orderivatives thereof. See, for example, Ditta et al., 1980 and EPA 0 120515. Alternatively, one may insert the sequences to be expressed inplant cells into a vector containing separate replication sequences, oneof which stabilizes the vector in E. coli, and the other inAgrobacterium. See, for example, McBride and Summerfeit 1990, whereinthe pRiHRI (Jouanin, et al., 1985), origin of replication is utilizedand provides for added stability of the plant expression vectors in hostAgrobacterium cells.

Included with the expression construct and the T-DNA is one or moreselectable marker coding sequences which allow for selection oftransformed Agrobacterium and transformed plant cells. A number ofantibiotic resistance markers have been developed for use with plantcells, these include genes inactivating antibiotics such as kanamycin,the aminoglycoside G418, hygromycin, or the like. The particular markeremployed is not essential to this invention, with a particular markerpreferred depending on the particular host and the manner ofconstruction.

For Agrobacterium-mediated transformation of plant cells, explants areincubated with Agrobacterium for a time sufficient to result ininfection, the bacteria killed, and the plant cells cultured in anappropriate selection medium. Once callus forms, shoot formation can beencouraged by employing the appropriate plant hormones in accordancewith known methods and the shoots transferred to rooting medium forregeneration of plants. The plants may then be grown to seed and theseed used to establish repetitive generations and for isolation of therecombinant protein produced by the plants.

There are a number of possible ways to obtain plant cells containingmore than one expression construct In one approach, plant cells areco-transformed with a first and second construct by inclusion of bothexpression constructs in a single transformation vector or by usingseparate vectors, one of which expresses desired genes. The secondconstruct can be introduced into a plant that has already beentransformed with the first expression construct, or alternatively,transformed plants, one having the first construct and one having thesecond construct, can be crossed to bring the constructs together in thesame plant.

In a preferred embodiment, the plants used in the methods of the presentinvention are derived from members of the taxonomic family known as theGramineae. This includes all members of the grass family of which theedible varieties are known as cereals. The cereals include a widevariety of species such as wheat (Triticum sps.), rice (Oryza sps.)barley (Hordeum sps.) oats, (Avena sps.) rye (Secale sps.), corn (maize)(Zea sps.) and millet (Pennisettum sps.). In practicing the presentinvention, preferred grains are rice, wheat, maize, barley, rye andtriticale, and most preferred is rice.

In order to produce transgenic plants that express human blood proteinin seeds, monocot plant cells or tissues derived from them aretransformed with an expression vector comprising the coding sequence fora human blood protein. The transgenic plant cells are cultured in mediumcontaining the appropriate selection agent to identify and select forplant cells which express the heterologous nucleic acid sequence. Afterplant cells that express the heterologous nucleic acid sequence areselected, whole plants are regenerated from the selected transgenicplant cells. Techniques for regenerating whole plants from transformedplant cells are generally known in the art Transgenic plant lines, e.g.,rice, wheat, corn or barely, can be developed and genetic crossescarried out using conventional plant breeding techniques.

Transformed plant cells are screened for the ability to be cultured inselective media having a threshold concentration of a selective agentPlant cells that grow on or in the selective media are typicallytransferred to a fresh supply of the same media and cultured again. Theexplants are then cultured under regeneration conditions to produceregenerated plant shoots. After shoots form, the shoots are transferredto a selective rooting medium to provide a complete plantlet. Theplantlet may then be grown to provide seed, cuttings, or the like forpropagating the transformed plants. The method provides for efficienttransformation of plant cells and regeneration of transgenic plants,which can produce a recombinant human blood protein.

The expression of the recombinant human blood protein may be confirmedusing standard analytical techniques such as Western blot, ELISA, PCR,HPLC, NMR, or mass spectroscopy, together with assays for a biologicalactivity specific to the particular protein being expressed.

A purified blood protein recombinantly produced in a plant cell,preferably substantially free of contaminants of the host plant cell,and preferably comprising at least one plant glycosyl group is alsoprovided by the invention. The plant glycosyl groups, while identifyingthat the blood protein was produced in a plant, does not significantlyimpair the biological activity of the blood protein in any of theapplied therapeutic contexts (preferably less than 25% loss of activity,more preferably less than 10% loss of activity, as compared to acorresponding non-recombinant human blood protein). Typically, inaccordance with some embodiments of the invention, the human bloodprotein constitutes at least about 0.5%, at least about 1.0% or at leastabout 2.0% of the total soluble protein in the seeds harvested from thetransgenic plant. In a preferred embodiment, however, protein expressionis much higher than previously reported, i.e., at least about 3.0%,which makes commercial production quite feasible. Advantageously,protein expression is at least about 5.0%, at least about 10%, at leastabout 15%, at least about 20%, at least about 30%, or even at leastabout 40% of total soluble protein.

The invention includes plant seed product prepared from the harvestedseeds. Preferably, the human blood protein constitutes at least about3.0% of the total soluble protein in the seed product, more preferablyat least about 5.0%, and most preferably at least about 10.0%. As shownin the figures, the expression of human blood proteins in rice grains,represented by AAT, the three fibrinogen polypeptides and HSA representat least about 10% of total soluble protein.

The present invention also provides compositions comprising human bloodproteins produced recombinantly in the seeds of monocot plants, andmethods of making such compositions. In practicing the invention, ahuman blood protein is produced in the seeds or grain of transgenicplants that express the nucleic acid coding sequence for the human bloodprotein. After expression, the blood protein may be provided to apatient in substantially unpurified form (Le., at least 20% of thecomposition comprises plant material), or the blood protein may beisolated or purified from a product of the mature seed (e.g., flour,extract, malt or whole seed, etc.) and formulated for delivery to apatient.

Such compositions can comprise a formulation for the type of deliveryintended. Delivery types can include, e.g. parenteral, enteric,inhalation, intranasal or topical delivery. Parenteral delivery caninclude, e.g. intravenous, intramuscular, or suppository. Entericdelivery can include, e.g. oral administration of a pill, capsule, orother formulation made with a non-nutritional pharmaceuticallyacceptable excipient, or a composition with a nutrient from thetransgenic plant, for example, in the grain extract in which the proteinis made, or from a source other than the transgenic plant Such nutrientsinclude, for example, salts, saccharides, vitamins, minerals, aminoacids, peptides, and proteins other than the human blood protein.Intranasal and inhalant delivery systems can include spray or aerosol inthe nostrils or mouth. Topical delivery can include, e.g. creams,topical sprays, or salves. Preferably, the composition is substantiallyfree of contaminants of the transgenic plant, preferably containing lessthan 20% plant material, more preferably less than 10%, and mostpreferably, less than 5%. The preferable route of administration isenteric, and preferably the composition is non-nutritional.

The blood protein can be purified from the seed product by a modeincluding grinding, filtration, heat, pressure, salt extraction,evaporation, or chromatography.

The human blood proteins produced in accordance with the invention alsoinclude all variants thereof, whether allelic variants or syntheticvariants. A “variant” human blood protein-encoding nucleic acid sequencemay encode a variant human blood protein amino acid sequence that isaltered by one or more amino acids from the native blood proteinsequence, preferably at least one amino acid substitution, deletion orinsertion. The nucleic acid substitution, insertion or deletion leadingto the variant may occur at any residue within the sequence, as long asthe encoded amino acid sequence maintains substantially the samebiological activity of the native human blood protein. In anotherembodiment, the variant human blood protein nucleic acid sequence mayencode the same polypeptide as the native sequence but, due to thedegeneracy of the genetic code, the variant has a nucleic acid sequencealtered by one or more bases from the native polynucleotide sequence.

The variant nucleic acid sequence may encode a variant amino acidsequence that contains a “conservative” substitution, wherein thesubstituted amino acid has structural or chemical properties similar tothe amino acid which it replaces and physicochemical amino acid sidechain properties and high substitution frequencies in homologousproteins found in nature (as determined, e.g., by a standard Dayhofffrequency exchange matrix or BLOSUM matrix). In addition, oralternatively, the variant nucleic acid sequence may encode a variantamino acid sequence containing a “non-conservative” substitution,wherein the substituted amino acid has dissimilar structural or chemicalproperties to the amino acid it replaces. Standard substitution classesinclude six classes of amino acids based on common side chain propertiesand highest frequency of substitution in homologous proteins in nature,as is generally known to those of skill in the art and may be employedto develop variant human blood protein-encoding nucleic acid sequences.

As will be understood by those of skill in the art, in some cases it maybe advantageous to use a human blood protein-encoding nucleotidesequences possessing non-naturally occurring codons. Codons preferred bya particular eukaryotic host can be selected, for example, to increasethe rate of human blood protein expression or to produce recombinant RNAtranscripts having desirable properties, such as a longer half-life,than transcripts produced from naturally occurring sequence. As anexample, it has been shown that codons for genes expressed in rice arerich in guanine (G) or cytosine (C) in the third codon position (Huanget al., 1990). Changing low G+C content to a high G+C content has beenfound to increase the expression levels of foreign protein genes inbarley grains (Horvath et al., 2000). The blood protein encoding genesemployed in the present invention were synthesized by OperonTechnologies (Alameda, C A based on the rice gene codon bias (Huang etal., 1990) along with the appropriate restriction sites for genecloning. These ‘codon-optimized’ genes were linked toregulatory/secretion sequences for seed-directed monocot expression andthese chimeric genes then inserted into the appropriate planttransformation vectors.

A human blood protein-encoding nucleotide sequence may be engineered inorder to alter the human blood protein coding sequence for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the human blood protein by acell.

Heterologous nucleic acid constructs may include the coding sequence fora given human blood protein (i) in isolation; (ii) in combination withadditional coding sequences; such as fusion protein or signal peptide,in which the human blood protein coding sequence is the dominant codingsequence; (iii) in combination with non-coding sequences, such asintrons and control elements, such as promoter and terminator elementsor 5′ and/or 3′ untranslated regions, effective for expression of thecoding sequence in a suitable host; and/or (iv) in a vector or hostenvironment in which the human blood protein coding sequence is aheterologous gene.

Depending upon the intended use, an expression construct may contain thenucleic acid sequence encoding the entire human blood protein, or aportion thereof. For example, where human blood protein sequences areused in constructs for use as a probe, it may be advantageous to prepareconstructs containing only a particular portion of the human bloodprotein encoding sequence, for example a sequence which is discovered toencode a highly conserved human blood protein region.

The invention provides, in one embodiment, a seed composition containinga flour, extract, or malt obtained from mature monocot seeds and one ormore seed produced human blood proteins in unpurified form. Isolatingthe blood proteins from the flour can entail forming an extractcomposition by milling seeds to form a flour, extracting the flour withan aqueous buffered solution, and optionally, further treating theextract to partially concentrate the extract and/or remove unwantedcomponents. In a preferred method, mature monocot seeds, such as riceseeds, are milled to a flour, and the flour then suspended in saline orin a buffer, such as Phosphate Buffered Saline (“PBS”), ammoniumbicarbonate buffer, ammonium acetate buffer or Tris buffer. A volatilebuffer or salt, such as ammonium bicarbonate or ammonium acetate mayobviate the need for a salt-removing step, and thus simplify the extractprocessing method.

The flour suspension is incubated with shaking for a period typicallybetween 30 minutes and 4 hours, at a temperature between 20-55° C., Theresulting homogenate is clarified either by filtration orcentrifugation. The clarified filtrate or supernatant may be furtherprocessed, for example by ultrafiltration or dialysis or both to removecontaminants such as lipids, sugars and salt. Finally, the materialmaybe dried, e.g., by lyophilization, to form a dry cake or powder. Theextract combines advantages of high blood-protein yields, essentiallylimiting losses associated with protein purification,

In general, the protein once produced in a product of a mature seed canbe further purified by standard methods known in the art, such as byfiltration, affinity column, gel electrophoresis, and other suchstandard procedures. The purified protein can then be formulated asdesired for delivery to a human patient More than one protein can becombined for the therapeutic formulation. The protein may be purifiedand used in biomedical applications requiring a non-food administrationof the protein,

The following examples illustrate but are not intended in any way tolimit the invention.

EXAMPLE 1

Production of Transgenic Rice Encoding AAT and Fibrinogen Polypeptides

The basic procedures of particle bombardment-mediated ricetransformation and plant regeneration were carried out as described byHuang et al., 2001, Rice variety TP309 seeds were dehusked, sterilizedin 50% (v/v) commercial bleach for 25 min. and washed with sterilewater. The sterilized seeds were placed on rice callus induction medium(RCI) plates containing [N6 salts (Sigma), B5 vitamins (Sigma), 2 mg/l2,4-D and 3% sucrose], The rice seeds were incubated for 10 days toinduce callus formation. Primary callus was dissected from the seeds andplaced on RCI for 3 weeks. This was done twice more to generatesecondary and tertiary callus which was used for bombardment andcontinued subculture, A callus of 1-4 mm diameter was placed in a 4 cmcircle on RCI with 0.3M mannitol 0.3M sorbitol for 5-24 hrs prior tobombardment. Microprojectile bombardment was carried out using theBiolistic PDC-1000/He system (Bio-Rad). The procedure requires 1.5 mggold particles (60 μg/ml) coated with 2.5 μg DNA. DNA-coated goldparticles were bombarded into rice calli with a He pressure of 1100psi.After bombardment, the callus was allowed to recover for 48 hrs. andthen transferred to RCI with 30 mg/l hygromycin B for selection andincubated in the dark for 45 days at 26° C. Transformed calli wereselected and transferred to RCI (minus 2,4-D) containing 5 mg/l ABA, 2mg/l BAP, 1 mg/l NAA and 30mg/l hygromycin B for 9-12 days. Transformedcalli were transferred to regeneration medium consisting of RCI (minus2,4-D), 3 mg/l BAP, and 0.5 mg/l NAA without hygromycin B and culturedunder continuous lighting conditions from 2-4 weeks. Regeneratedplantlets (1-3 cm high) were transferred to rooting medium whoseconcentration was half that of MS medium (Sigma) plus 1% sucrose and0.05 mg/l NAA. After 2 weeks on rooting medium, the plantlets developedroots and the shoots grew to about 10 cm. The plants were transferred toa 6.5×6.5 cm pots containing a mix of 50% commercial soil (Sunshine #1)and 50% soil from rice fields. The plants were covered by a plasticcontainer to maintain nearly 100% humidity and grown under continuouslight for 1 week. The transparent plastic cover was slowly shifted overa 1 day period to gradually reduce humidity and water and fertilizersadded as necessary. When the transgenic RO plants were approximately 20cm in height, they were transferred to a greenhouse where they grew tomaturity.

Individual R1 seed grains from the individual R0 regenerated plants weredissected into embryos and endosperms. Expression levels of recombinantblood proteins (AAT and fibrinogen polypeptides) in the isolated riceendosperms were determined. Embryos from the individual R1 grains withhigh recombinant protein expression were sterilized in 50% bleach for 25min and washed with sterile distilled water. Sterilized embryos wereplaced in a tissue culture tube containing ½ MS basal salts with theaddition of 1% sucrose and 0.05 mg/l NAA. Embryos were germinated andplantlets having ˜7 cm shoots and healthy root systems were obtained inabout 2 weeks. Mature R1 plants were obtained as regenerants.

EXAMPLE 2

Production of Rice Extract Containing Recombinant Blood Proteins and itsUse in Parenteral and Enteric Formulations General Procedure forProduction of Rice Extract

Transgenic rice containing heterologous polypeptides can be converted torice extracts by either a dry milling or wet milling process. In the drymilling process, transgenic paddy rice seeds containing the heterologouspolypeptides were dehusked with a dehusker. The rice was grounded into afine flour though a dry milling process, for example, in one experiment,at speed 3 of a model 91 Kitchen Mill from K-TEC. Phosphate bufferedsaline (“PBS”), containing 0.135 N NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.7mM KH₂PO₄, at pH 7.4, with or without additional NaCl, such as 0.35 NNaCl, was added to the rice flour. In some experiments, approximately 10ml of extraction buffer was used for each 1 g of flour. In otherexperiments, the initial flour/buffer ratio varied over a range such as1 g/40 ml to 1 g/10 ml. The mixture was incubated at room temperaturewith gentle shaking for 1 hr. In other experiments, the incubationtemperature was lower or higher, such as from about 22° C. to about 60°C., and the incubation time was longer or shorter, such as from about 10minutes to about 24 hours. A Thermolyne VariMix platform mixer set athigh speed was used to keep the particulates suspended.

In place of PBS, other buffers such as ammonium bicarbonate, were usedin some experiments. In one embodiment, 10 liters of 0.5M ammoniumbicarbonate was added to 1 kg of rice flour.

The resulting homogenate was clarified either by filtration orcentrifugation. For the filtration method, the mixture was allowed tosettle for about 30 minutes at room temperature, after which thehomogenate was collected and filtered. Filters in three differentconfigurations were purchased from Pall Gemansciences and used. Theywere: a 3 μm pleated capsule, a 1.2 μm serum capsule and a Suporcapcapsule 50 (0.2 μm). For centrifugation, a Beckman J2-HC centrifuge wasused and the mixture was centrifuged at 30,000 g at 4° C. for about 1hour. The supernatant was retained and the pellet discarded.

In one embodiment, the filtrate and supernatant were further processed,for example by ultra-filtration or dialysis or both to remove componentssuch as lipids, sugars and salt.

The filtrate from the above filtration procedure, which is also calledthe clarified extract, was then concentrated using a spiral woundtangential flow filter operated in a batch recirculation mode. In oneembodiment, PES (polyethersulfone) 3000-4000 molecular weight cutoffmembranes were used for this step. These final concentrated extractswere held overnight in a cold room.

The concentrated extracts were next dried to a powder by lyophilization.The lyophilized material was scraped from the lyophilizer trays andcombined into a plastic bag. The dry material was compressed by drawinga vacuum on the bag and then the material was blended and the particlesize reduced by hand-kneading it through the plastic.

Rice extract can also be produced using a wet milling procedure.Transgenic paddy rice seeds containing recombinant human blood proteincan be rehydrated for a period of 0 to 288 hrs at 30° C. The rehydratedseeds are ground in PBS extraction buffer. The initial seed/buffer ratiocan vary over a range such as 1 g/40 ml to 1 g/10 ml.

Over 20% human blood protein can be recovered from the wet millingprocess. The result of the wet milling becomes an initial extract thatmay be kept cold (4° C.) or stored frozen until use depending on thestability of the blood protein target The processing of initial extractto obtain dried extract is the same as that described for dry milling inthis section.

EXAMPLE 3

Concentration and Diafiltration of Recombinant Blood Protein and ControlRice Extracts.

The conditions used in concentration and diafiltration vary depending onvolume, speed, cost, etc. These conditions are standard in the art basedon the description herein. The frozen initial extract was thawed in thecoldroom (about 2-8° C.) for six hours. The thawed material wasclarified though a 0.45 μm filter and concentrated using a 5000 NominalMolecular Weight Cutoff membrane of Polyethersultone.

90 ml of the filtrate of control extract was concentrated to 10 ml andadditional 10 ml of deionized water can be added to the concentratedfiltrate. The diluted filtrate can be diafiltrated one more time usingwater. The precipitate starts forming at 16 mS and increases as theionic strength decreases. A solution of 1.0M ammonium bicarbonate wasadded to the retentate to add ionic strength. The haze decreasesalthough does not disappear completely. The material was diafilteredmultiple times, in one embodiment three times, with water and multipletimes, in one embodiment three times, with 0.1 M ammonium bicarbonate.It was concentrated to 9 ml and the membrane is rinsed with 0.1 Mammonium bicarbonate. The concentrate was filtered through several 0.211 m button filters. In one embodiment, 2.3 ml of the filtrate islyophilized as is; 2.3 ml of the filtrate is diluted to 12 ml withdeionized water and lyophilized, and 2.0 ml of the filtrate is dilutedto 25 ml with deionized water and lyophilized. All the filtratesremained clear.

A total of 89 ml of the filtrate of recombinant protein extract wasconcentrated to 10 ml, and additional 10 ml of 0.1 M ammoniumbicarbonate is added. The resulting mixture is concentrated back to 10ml and another 10 ml of 0.1 M ammonium bicarbonate is added. Theretentate starts to haze up. The material was diafiltered multipletimes, in one embodiment three times, with 0.1 M ammonium bicarbonate.It was concentrated to 9 ml and the membrane is rinsed with 0.1 Mammonium bicarbonate. The concentrate was filtered through several 0.45μm button filters. In one embodiment, 2.0 ml of the filtrate waslyophilized as is; 2.0 ml of the filtrate was diluted to 12 ml withdeionized water where a haze formed, and lyophilized, and 2.0 ml of thefiltrate was diluted to 12 ml with 0.1 M ammonium bicarbonate thatremained clear, and lyophilized.

EXAMPLE 4

Comparison of Trial Extraction of Recombinant Protein Rice with PBS andAmmonium Bicarbonate

The conditions used in concentration and diafiltration vary depending onvolume, speed, cost, etc. These conditions are all standard in the artbased on the description herein. Recombinant protein rice flour is mixedwith extraction buffer at about 100 g/L for about 1 hour using amagnetic stir bar. In one 2 L beaker, the extraction buffer is PBS, pH7.4 plus 0.35 M NaCl. In another 2 L beaker, the extraction buffer is0.5 M ammonium bicarbonate. A 15 cm Buchner funnel is precoated withabout 6 g of Cel-pure C300 before adding another 20 g of Cel-pure C300.The mixture is filtered at about 3-4 Hg. It is then washed twice withabout 100 nil of respective extraction buffer. The extracted filtrate iscollected and concentrated with ultra-filtration cartridges: 5KRegenerate Cellulose, 5K PES, and 1K Regenerated Cellulose. Theconcentrates are lyophilized and analyzed for recombinant blood proteinactivity contents. The ammonium bicarbonate and PBS, pH 7.4 plus 0.35 MNaCl both extract approximately the same amount of rAAT. There is littleloss of recombinant protein units in the permeate with any of theultrafiltration units that were used,

Other extraction buffer can also be used to extract recombinant proteinsexpressed in transgenic rice grains, for example Tris buffer, ammoniumacetate, depending on applications.

EXAMPLE 5

Production of Rice Extracts Containing Recombinant Blood Proteins

The conditions used in concentration and diafiltration vary depending onvolume, speed, cost, etc, These conditions are all standard in the artbased on the description herein, All equipment is soaked in hot 0.1MNaOH at a starting temperature of about 55° C. Rice flour is added to anabout 250-500 gal stainless steel tank containing 0.5M ammoniumbicarbonate in a ratio of 95-105 g/L. It is mixed for about 60-80minutes at about 9° C.

12 plates of 36 inch filter press C300 were pre-coated with about 3-6 kgCel-pure C300. About 19-26 g/L of Cel-pure is added to the extract andmixed thoroughly. The mixture is pressed at a pressure of about 22 psiat a flow rate of about 82 liters/minute. The filtrate is collected intoa 250 gal stainless steel tank and washed with 0.5M ammoniumbicarbonate. The press is blown dry. This process is carried out atabout 10° C.

The 300 NMW cut-off membranes (Polysulfone), which had been cleaned andstored with 0.1M NaOH after control run is rinsed thoroughly withdeionized water. The extract is concentrated and bumped to a 100 gallonstainless steel tank. The membrane and the concentration tank wereflushed with 0.1 M ammonium bicarbonate to recover all remainingextract. The products were covered with plastic and left in the 100gallon tank overnight at room temperature. The concentrate is filteredthrough a spiral wound 1 μm filter and into a 5 gallon poly container.

EXAMPLE 6

Blending of Rice Extract Containing Recombinant Proteins intoParenteral, Inhalant, Intranasal and Topical Formulations.

Recombinant blood proteins (such as AAT) can be highly purified grainsfrom cereal grains for use in medical/pharmaceutical applications. Apurification protocol for rice seed extract expressed human AAT has beendeveloped [Huang et al., 2002], consisting of preparing a rice seedextract according to the above examples and further purifying theextract preparation using Con-A, DEAE and Octyl Sepharose chromatographyrespectively. AAT can be purified to greater than 90% homogeneityutilizing such a procedure [Huang et al., 2002]. Purified AAT can beutilized in potential pharma/medical applications for the followingindications: AAT augmentation/replacement therapy [Sandhaus, 1993;Hubbard et al., 1989], cystic fibrosis [McElvaney et al., 1991;Allen,1996], psoriasis, panniculitis and cutaneous vasculitis [O'Riordanof al., 1997; Dowd et al., 1995] and pulmonary inflammation [Bingle andTetley, 1996]. For some of these indications, purified AAT proteinpreparations can be administered via intravenous (iv) methods in 0.09%saline solution. Alternatively, the saline solution could be bufferedwith serum albumen at 0.5% or some other pharmacologically acceptableprotein carrier molecule. AAT dosages are usually around 60 mg/kg. Foraerosol delivery, an aerosol generating system can be employed utilizinga compressed air driven nebulizer selected on the basis of the basis ofits ability to generate an aerosol with droplets of the optimum size (<3um in aerodynamic diameter) for deposition in the lower respiratorytract [Hubbard of al., 1989). Again proteins can either be suspended insterile water or a buffered saline solution containing apharmacologically acceptable protein carrier. Alternatively, a driedprotein powder containing the purified protein component could beutilized as the dispersal agent and this could be an a rice basedextract where the AAT component is greater but not less then 50% byweight.

In another case, recombinant rice expressed and extracted human bloodproteins such as AA T and fibrinogen can be employed topically. The useof fibrin sealants/bandages has been a widely accepted used by themedical community. Fibrin sealants are effective hemostatic agents[Mankad and Codispoti, 2001], a means for achieving tissue adhesion,preventing fluid accumulation and promotion of wound healing [Spotnitz,2001]. Fibrin sealants can also be used as a means of slowly releasingmedications, including antibiotics, growth factors and other agents[Spotnitz, 1997]. Rice expressed fibrinogen can also provide a potentiallow cost and animal virus free source for these indications.

What is claimed is:
 1. A method of producing a recombinant human bloodprotein in monocot plant seeds, comprising the steps of: (a)transforming a monocot plant cell with a chimeric gene comprising (i) apromoter from the gene of a maturation-specific monocot plant storageprotein operably linked to a first DNA sequence encoding a monocot plantseed-specific signal sequence capable of targeting a polypeptide linkedthereto to a monocot plant seed endosperm cell, wherein the promoteroperably linked to the sequence encoding the signal sequence comprisesSEQ ID NO: 7; and (ii) a second DNA sequence encoding the human bloodprotein, linked in translation frame with the first DNA sequence,wherein the first DNA sequence and the second DNA sequence togetherencode a fusion protein comprising an N-terminal signal sequence and thehuman blood protein; (b) growing a monocot plant from the transformedmonocot plant cell for a time sufficient to produce seeds containing thehuman blood protein; and (c) harvesting the seeds from the plant,wherein the human blood protein constitutes at least about 3.0% of thetotal soluble protein in the harvested seeds.
 2. The method of claim 1,wherein the human blood protein is selected from the group consisting ofhemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin (HSA),thrombin, antithrombin Ill, an antibody, a growth factor and a bloodcoagulation factor.
 3. The method of claim 1, wherein the human bloodprotein constitutes at least about 5.0% of the total soluble protein inthe harvested seeds.
 4. The method of claim 1, wherein the human bloodprotein constitutes at least about 10.0% of the total soluble protein inthe harvested seeds.
 5. The method of claim 1, further comprisingpurifying the human blood protein from the harvested seeds.
 6. Themethod of claim 1, wherein the human blood protein produced in themethod comprises one or more plant glycosyl groups.
 7. A compositioncomprising a purified recombinant human blood protein obtained by themethod of claim 1, wherein the human blood protein comprises one or moreplant glycosyl groups; and wherein said composition comprises SEQ ID NO:7.
 8. The composition of claim 7, wherein the human blood protein isselected from the group consisting of hemoglobin, alpha-1-antitrypsin,fibrinogen, human serum albumin (HSA), thrombin, antithrombin III, anantibody, a growth factor and a blood coagulation factor.
 9. A monocotplant seed product selected from the group consisting of whole seed,flour, extract, malt, and a partially purified protein fraction,prepared from the harvested seeds obtained by the method of claim 1,wherein the human blood protein constitutes at least about 3.0% of thetotal soluble protein in the seed product; and wherein the seed productcomprises SEQ ID NO:
 7. 10. The seed product of claim 9, wherein thehuman blood protein constitutes at least about 5.0% of the total solubleprotein in the seed product.
 11. The seed product of claim 9, whereinthe human blood protein constitutes at least about 10.0% of the totalsoluble protein in the seed product.
 12. The seed product of claim 9,wherein the human blood protein is selected from the group consisting ofhemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin (HSA),thrombin, antithrombin III, an antibody, a growth factor and a bloodcoagulation factor.
 13. A composition comprising: a purified recombinanthuman blood protein obtained by the method of claim 1, wherein the humanblood protein comprises one or more plant glycosyl groups; and at leastone pharmaceutically acceptable excipient or nutrient from a sourceother than the monocot plant, wherein the excipient or nutrient isselected from the group consisting of salts, saccharides, vitamins,minerals, amino acids, peptides and proteins other than the human bloodprotein; wherein said composition comprises SEQ ID NO:
 7. 14. Thecomposition of claim 13, wherein the human blood protein is selectedfrom the group consisting of hemoglobin, alpha-1-antitrypsin,fibrinogen, human serum albumin (HSA), thrombin, antithrombin Ill, anantibody, and a blood coagulation factor.
 15. The composition of claim13, wherein the composition is formulated for parenteral, enteric,inhalation, intranasal or topical delivery.
 16. The composition of claim13, wherein the composition is formulated for enteric delivery.